This invention is in the field of chemical compounds comprising steroid and poly-L-lysine moieties. Such compounds may also be complexed with nucleic acids and are useful for targeting biologically active materials to prostate cell nuclei for example for gene therapy, or cancer therapy or diagnosis.
Prostate cancer is the most commonly diagnosed cancer in American males, with an expected incidence of 200,000 in 1994. The projected deaths from prostate cancer are 38,000 in the same year. No curative therapy exists for advanced or metastatic prostate cancer. Chemotherapy is ineffective. Androgen ablation is palliative and non-curative. The molecular characterization of the disease has progressed very rapidly in the past few years, and it now appears that gene therapy will be the treatment of choice for advanced and metastatic disease within ten years. Several potential genetic targets of therapy exist including replacement of tumor suppressor genes, or metastasis suppressor genes, shutdown of oncogenes, and genetic induction of programmed cell death (apoptosis).
Prostate cancer is a genetically diverse disease. Some prostate cancers over-express the Ras oncoprotein, but not all. Approximately half of the prostate cancers fail to express retinoblastoma (RB) mRNA (Petros, J. A. et al. [1991], xe2x80x9cInvestigation of retinoblastoma transcripts in primary prostatic adenocarcinoma,xe2x80x9d J. Urology 145:293A; Bookstein, R., et al. [1990], xe2x80x9cIntroduction of a normal retinoblastoma gene [RB] into retinoblastoma,xe2x80x9d Science, 247:712-715). Absent or abnormal RB transcripts will result in lack of protein expression and lack of normal regulation of cell cycle.
Some prostate cancers show the common p53 promoter mutation (Bookstein, R. et al. [1993], xe2x80x9cp53 is mutated in a subset of advanced-stage prostate cancers,xe2x80x9d Cancer Res. 53:3369-3373), but most do not. This genetic diversity is consistent with the well-accepted xe2x80x9cmulti-hitxe2x80x9d theory of solid tumor oncogenesis, and is responsible for the varied clinical manifestations of the disease. This genetic diversity is also responsible for the hormonal responsiveness of some tumors and the hormonal resistance of others.
Correction of mutant RB or p53 stops tumor growth. The RB gene was shown to be mutant in a prostate cancer cell line, and the transfection of wild-type RB shown to abrogate tumorigenicity in nude mice (Bookstein, R., et al. [1990], xe2x80x9cSuppression of Tumorigenicity of Human Prostate Carcinoma Cells by Rephasing a Mutated RB Genexe2x80x9d Science, 247:712-715) and in human tumor cells. (Chen, P-L, et al. [1990], xe2x80x9cGenetic Mechanisms of Tumor Suppression by the Human p53 Gene,xe2x80x9d Science 250:1576-1580). Subsequent reports which indicated that this was an uncommon event in primary human tumors (Isaacs, W. B., et al. [1991], xe2x80x9cWild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles,xe2x80x9d Cancer Res. 51:4716-4720) were flawed in assaying for only one mutation. Studies in our laboratories have demonstrated that in fact up to fifty percent of tumors fail to make normal RB transcripts (Petros, J. A. et al. [1991], xe2x80x9cInvestigation of retinoblastoma transcripts in primary prostatic adenocarcinoma,xe2x80x9d J. Urology 145:293A), and it is likely that an even greater percentage will be shown to be abnormal when large numbers of tumors have been assayed for protein expression. Similarly, p53 has been shown to be mutated in twenty to twenty-five percent of advanced-stage prostate cancers (Bookstein, R. et al. (1993), xe2x80x9cp53 is mutated in a subset of advanced-stage prostate cancers,xe2x80x9d Cancer Res. 53:3369-3373), but relatively few early stage prostate cancers. The reintroduction of p53 into cell lines with mutant p53 abolished their ability to grow and divide (Isaacs, W. B., et al. [1991], xe2x80x9cWild-type p53 suppresses growth of human prostate cancer cells containing mutant p53 alleles,xe2x80x9d Cancer Res. 51:4716-4720).
A further tumor suppressor gene is p16 (also known as Multiple Tumor Suppressor 1 (MTS1), which encodes the p16 inhibitor of cyclin-dependent kinase 4 (Kamb, A., et al. [1994], xe2x80x9cA cell cycle regulator potentially involved in genesis of many tumor types,xe2x80x9d Science 264 436-440; Nobori, T., et al. [1994], xe2x80x9cDeletions of the cyclin-dependent kinase-4 inhibitor gene in multiple human cancers,xe2x80x9d Nature 368:753-756). DNA encoding genes for suppression of metastasis of prostate cancer is also known to the art, and has been used via microcell-mediated chromosome transfer to suppress metastatic ability of microcell hybrids (Ichikawa, T., et al., xe2x80x9cSuppression of metastasis of rat prostatic cancer by introducing human chromosome 8,xe2x80x9d Cancer Res. 54:2299-2302).
Polylysine DNA complexes have been used to transfer genes into cells in vitro (Curiel, D. T., et al. (1991), xe2x80x9cAdenovirus enhancement of transferrin-polylysine-mediated gene delivery,xe2x80x9d Proc. Natl. Acad. Sci. USA 88:8850-8854; PCT application PCT/EP92/02234 for xe2x80x9cComposition for Introducing Nucleic Acid Complexes Into Higher Eukaryotic Cells,xe2x80x9d claiming priority to U.S. application Ser. No. 07/937,788 to Curiel, et al. filed Sep. 2, 1992, which is fully incorporated herein by reference) and in vivo (Gao, L., et al. [1993], xe2x80x9cDirect In Vivo Gene Transfer to Airway Epithelium Employing Adenovirus-polylysine-DNA complexes,xe2x80x9d Human Gene Therapy 4:17-24).
The surface receptor for asialoorosomucoid has been found to mediate DNA uptake in hepaitocytes. A soluble DNA carrier system consisting of an asialoglycoprotein linked to poly-L-lysine has been used to bind DNA and hepatitis B virus DNA constructs to liver cells. Liver cells express specific surface receptors for asialoorosomucoid. Covalent linkage of asialoorosomucoid with poly-L-lysine followed by ionic bonding with DNA creates a soluble delivery system (Wu, G. Y. and Wu, C. H. [1987], xe2x80x9cReceptor-mediated in vitro gene transformation by a soluble DNA carrier,xe2x80x9d J. Biol. Chem. 262:4429-4432). The same asialoorosomucoid-poly-L-lysine-DNA construct has been used to selectively transform the liver in vivo in a rat model. (Wu, G. Y. and Wu, C. W. [1988], xe2x80x9cReceptor-mediated Gene Delivery and Expression in Vivo,xe2x80x9d J. Biol. Chem. 368:14621-14624). Transformation of asialoglycoprotein receptor-positive human hepatoma cells with this system has also has also been shown. (Liang T. J., et al. [1993], xe2x80x9cTargeted transfection and expression of hepatitis B viral DNA in human hepatoma cells,xe2x80x9d J. Clin. Invest. 91:1241-1246). Using this receptor-mediated delivery and targeting system it has been possible to induce production of proteins encoded for by the DNA so introduced. This has been shown to be receptor mediated since competitive binding with non-linked asialoorosomucoid abrogated the expression. In addition, receptor-negative cells do not take up the DNA or express the proteins. After intravenous injection, DNA complexed with asialoglycoprotein-polylysine conjugates is expressed transiently. Cytoplasmic vesicles are the main site of persistence of endocytosed DNA (Chowdhury, N. R., et al. [1993], xe2x80x9cFate of DNA Targeted to the Liver by Asialoglycoprotein Receptor-mediated Endocytosis in Vivo,xe2x80x9d J. Biol. Chem. 268:11265-11271).
Transferrin-polycation complexes (transferrin-polylysine and transferrin-protamine) have been used to transfer reporter genes into hematopoietic cells (Zenke, M., et al. [1990], xe2x80x9cReceptor-mediated endocytosis of transferrin-polycation conjugates: an efficient way to introduce DNA into hematopoietic cells,xe2x80x9d Proc. Natl. Acad. Sci. U S A, 87:3655-3659; Wagner, et al. [1990], xe2x80x9cTransferrin-polycation conjugates as carriers for DNA uptake into cells,xe2x80x9d Proc. Natl. Acad. Sci. U S A 87:3410-3414). (A transferrin-poly-L-lysine is commercially available as hT fpL/AdpL of Serva Biochemical and was used in combination with antibody-bound adenovirus to improve efficiency of endocytosis in HeLa cells in culture (Michael, S. L. et al., [1993] xe2x80x9cBinding-incompetent Adenovirus Facilitates Molecular Conjugate-Mediated Gene Transfer by the Receptor-mediated Endocytosis Pathway,xe2x80x9d J. Biol. Chem. 268:6866-6869).
PCT Application WO 91/177 3 published November 28, 1991 to Boehringer Ingelheim, incorporated herein by reference, relates to a system for transporting nucleic acids with a specific activity for T-cells. This system makes use of cell surface proteins of the T-cell lineage, e.g. CD4, the receptor used by the HIV virus. The nucleic acid to be imported is complexed with a protein-polycation conjugate, the protein component of which, i.e. the recognition domain, is a protein capable of binding to the T-cell surface protein, e.g. CD4, and cells which express this surface protein are brought into contact with the resulting protein-polycation/nucleic acid complexes. It has been shown that DNA transported into the cell by means of this system is expressed in the cell.
Steroid hormones interact with components of biological membranes and may enter their respective target cells by diffusion or by a membrane-mediated process which is saturable and temperature-dependent (Pietras, R. J. and Szego, C. M. [1977], xe2x80x9cSpecific binding sites for oestrogen at the outer surfaces of isolated endometrial cells,xe2x80x9d Nature 265:69-72). The subcellular distribution of steroid receptors in target tissues is still a controversial issue. Little information has been gathered concerning the nature and properties of membrane-bound steroid receptors. Plasma membranes of target cells may contain steroid receptors (Steinsapir, J. and Muldoon, T. G. [1991], xe2x80x9cRole of microsomal receptors in steroid hormone action,xe2x80x9d Steroids 56:66-71).
The human androgen receptor has been characterized and consists of a protein comprising 917 amino acids having three domains that regulate transcription, DNA binding, and steroid specificity (Coffey, O. S. [1992], xe2x80x9cThe molecular biology, endocrinology, and physiology of the prostate and seminal vesicles,xe2x80x9d In Campbell""s Urology, 6th Ed [Walsh, P. C. et al. eds.] Philadelphia, W. B. Saunders Co., p. 242). The androgen receptor may be found in prostate tissue, metastatic cancerous prostate tissue, hair follicles, muscle, and skin. Androgen receptors are cytoplasmic but may also be located in the cell membrane. A specific mechanism, associated with the cell membrane, must transport steroids into the target cell before they can bind to the cytosolic receptor (Harrison, R. W., et al. [1974], xe2x80x9cEvidence for Glucocorticoid Transport through the Target Cell Membrane,xe2x80x9d Biochem. Biophys. Res. Comm. 61:1262-1267).
Androgen receptors have affinity for a number of androgens and other steroids. Prostate tumor cells have affinity for progestagenic and estrogenic steroids (Veldscholte, J., et al. [1990], xe2x80x9cUnusual specificity of the androgen receptor in the human prostate tumor cell line LNCaP: high affinity for progestagenic and estrogenic steroids,xe2x80x9d Biochimica et Biophysica Acta. 1052:187-194). Such tumor cells also have affinity to antiandrogens such as cyproterone acetate (Veldscholte, J., et al. [1992], xe2x80x9cThe Androgen Receptor in LNCaP Cells Contains a Mutation in the Ligand Binding Domain which Affects Steroid Binding Characteristics and Response to Antiandrogens,xe2x80x9d J. Steroid Biochem. Molec. Biol. 41:665-669). Androgen deprivation results in increased expression of the androgen receptor. Fourfold increase in androgen receptor mRNA in the prostate followed medical castration or rats (Blok, L. J., et al. [1992], xe2x80x9cEffect of testosterone deprivation on expression of the androgen receptor in rat prostate, epididymis and testis,xe2x80x9d Int. J. of Andrology 15:182-198).
Effective gene therapy for metastatic disease depends upon delivery of a high concentration of the therapeutic agent to the sites of metastases. This delivery is difficult with respect to prostate tissue because of the plasma solubility requirements of agents. Uptake of therapeutic agents by prostate tissue has generally been thought to require lipophilic carriers.
There is a need for an efficient water-soluble system for introducing nucleic acid into androgen receptor-containing cells, especially prostate cells and metastatic prostate cells, especially for purposes of gene therapy and cancer therapy.
This invention provides a water-soluble delivery system for nucleic acids to cells having androgen receptors, preferably prostate cells, including cancerous or metastatic prostate cells. The invention includes a compound comprising a steroid moiety capable of binding to an androgen receptor, said steroid moiety being covalently linked to a polycationic salt. This compound is useful for complexing with nucleic acids desired to be delivered to the cells. The compounds are useful for gene therapy, cancer therapy, and diagnosis. The use of a moiety for which the androgen receptor has affinity enables targeting of injectable materials to prostate and other androgen receptor-containing cells. Such injectable materials must be water-soluble (serum-soluble). Previously it has been believed that compounds must be provided in lipophilic carriers in order to be taken up by prostate cells.
Many steroid moieties are capable of binding to androgen receptors including androgens, estrogens, synthetic androgens and estrogens, antiandrogens, metabolically altered analogs of the foregoing, and others, all as known to the art.
Polycationic salts useful for completing with nucleic acids include salts of cationic polyamines such polylysines, specifically poly-L-lysines, polyarginines, specifically poly-L-arginine, polyhistidine, and protamines.
The counter-ion of such salts can be any pharmaceutically acceptable ion, preferably a halogen ion, and most preferably bromide.
This invention also provides a method for expressing a protein in a cell containing androgen receptors comprising contacting said cell with a nucleic acid complex of a compound comprising a steroid moiety capable of binding to an androgen receptor of said prostate cell, the steroid moiety being covalently linked to a polycationic salt having a pharmaceutically acceptable counterion, wherein said nucleic acid encodes said protein, whereby said protein is detectably expressed in said cell. Preferably said prostate cell is a cancerous cell, which term includes metastatic prostate cells not localized to the prostate.
A method is also provided for targeting a biologically active material to the nucleus of a cell containing an androgen receptor comprising contacting said cell with a composition comprising said biologically active material covalently or ionically bonded to a compound comprising a steroid moiety covalently linked to a polycationic material whereby said biologically active material is delivered to the nucleus of said cell.
When the foregoing methods comprise injecting the complex into a suitable host, dosages in the range of from about 5 mg to about 250 mg DNA complexed with about 5 to about 10 mg steroid/polycationic compound are suitable.
This invention also provides a method for detecting the location of androgen-receptor containing cells in a subject comprising injecting into said subject a pharmaceutically effective amount of a nucleic acid complex of a compound comprising a steroid moiety capable of binding to an androgen receptor of said prostate cell, said steroid moiety being covalently linked to a polycationic salt having a pharmaceutically acceptable counterion, wherein said nucleic acid comprises detectably labelled nucleic acid. Dosages in the range of from about 5 mg to about 250 mg DNA completed with about 5 to about 10 mg steroid/polycationic compound are suitable for diagnosis and are capable of identifying the sites of metastatic tissue.
This invention involves the use of compounds containing steroids capable of binding to androgen receptors for targeting therapeutic or diagnostic molecules to prostate cells and other cells containing androgen receptors. Such therapeutic compounds are used for gene therapy and for treatment of prostate cancers. Diagnostic molecules include radiolabelled nucleic acids to diagnose metastatic prostate cancer sites.
Steroids have the following general structure: 
R is generally O or OH. A line above C-10 or C-13 denotes a methyl group. Substituents above the plane are termed beta- oriented and shown by a solid bond. In contrast, a substituent that is below the plane is alpha-oriented and denoted by a dashed line. A hydrogen atom attached to C-5 can be alpha or beta-oriented. When this hydrogen atom is alpha-oriented, the A and B rings are fused in a trans conformation, whereas a beta-orientation yields a cis fusion. The absence of a symbol for the C-5 hydrogen atom implies a trans fusion. The C-5 hydrogen atom is alpha-oriented in all naturally-occurring steroid hormones that contain a hydrogen atom in that position. A trans fusion yields a nearly planar structure, whereas a cis fusion gives a buckled structure.
Preferably the steroid moiety contains unsaturation in the A and/or B rings and has a substantially planar structure.
Some steroid moieties which bind to androgen receptors and are suitable for use in the compounds of this invention are: dihydrotestosterone, testosterone, estradiol, progesterone, androstanediol, androstenedione, hydroxyandrostenedione, mibolerone, cortisol, methyltrienolone, promegestone, triamcinolone acetonide, cyproterone acetate, hydroxyflutamide, nilutamide, casodex, tamoxifen, and 17xcex2-hydroxy-17xcex1-methyl-estra-4,9,11-trien-3-one (R1881). Most preferably the steroid moiety is dihydrotestosterone. Relative binding affinities of several steroids for the androgen receptor of LNCaP prostate tumor cells are given by Veldscholte, J. et al. (1990), xe2x80x9cUnusual specificity of the androgen receptor in the human estrogenic steroids,xe2x80x9d Biochemica et Biophysica Acta, 1052:187-194, as follows:
The polycationic material is covalently bonded to the steroid moiety at a site which does not interfere with binding of the molecule to an androgen receptor. Preferably, the polycationic material is attached at C-20. The covalent bond may be any covalent bond known to the art. Preferably the linkage is an ester bond formed by condensation of a C-20 alcohol with a carboxyl moiety of said polycationic material. More preferably, the linkage is an amide linkage formed by condensation of a C-20 amine with a carboxyl moiety of said polycationic material, or a disulfide bond.
The polycationic material is preferably poly-L-lysine, and may be of any molecular weight range. The invention is exemplified using a poly-L-lysine of 59 kD to complex with 7.4 kb of DNA. However, as will be appreciated by those skilled in the art, any size polycationic material may be used provided the molecule is of sufficient size to complex the desired nucleic acid. The charge of the polycationic material may also be adjusted to ensure complexing of the desired nucleic acid. The complexing may be done as exemplified herein or by any means known to the art.
A preferred embodiment is a dihydrotestosterone moiety covalently linked via an ester linkage to poly-L-lysine hydrobromide of the following formula: 
xe2x80x9cnxe2x80x9d can be any number greater than zero and preferably equals about 100 to about 1,000, and more preferably equals about 500 to about 600.
In a preferred embodiment, the steroid/polycationic compound of this invention is complexed with nucleic acid. The nucleic acid is preferably one which is capable of therapeutically modifying or locating a prostate cell. The prostate cell may be normal or abnormal, e.g. may be a tumor or metastatic cell, or a normal prostate cell. Preferably the nucleic acid is DNA, as it is less resistant to degradation than RNA when injected.
As will be readily appreciated by those skilled in the art, the complex can also contain other moieties such as adenovirus and adenovirus antibody sequences to aid endocytosis and help prevent degradation. Liposomes may also be used as part of the delivery systems of this invention.
This androgen receptor mediated delivery system, i.e., the steroid/polycationic molecule of this invention, targets therapeutic or diagnostic material to the nucleus as the specific subcellular site and therefore overcomes previous problems with trapping the material in non-nuclear locations.
The nucleic acid complexes of this invention are useful for gene therapy for prostate cancer. This may be accomplished by targeting a normal tumor or metastasis suppressor gene to a prostate cell lacking the normal suppressor gene. Suitable suppressor genes for this purpose are retinoblastoma (RB), p53, p16, and prostate cancer metastasis suppressor, all known to the art as discussed above. Prefer ably the suppressor gene is RB or p53.
Over-expression of the Ras gene is also a defect which may be corrected by gene therapy, such as by complexing inhibitors such as antisense DNA to the gene or promoter to the targeting compound.
Nucleic acids as therapeutically effective substances can also be used to inhibit specific cell functions, e.g. antisense RNAs and DNAs have proved effective in the selective inhibition of specific gene sequences. Their mode of activity enables them to be used as therapeutic agents for blocking the expression of certain genes (such as deregulated oncogenes or viral genes) in vivo. When the nucleic acid complexes of this invention contain prostate-specific antigens, antisense DNA inhibiting regulation and growth of normal cells can also be included in the complexes.
As not all prostate cancers have defects in suppressor genes, any highly successful therapeutic protocol for advanced prostate cancer must take this into account. Therapy must be individualized, and based on a meticulous characterization of each patient""s cancer, its genetic composition, and phenotypic behavior. This is particularly true for strategies involving gene therapy. It does no good to transfect the RB or p53 cDNA into a patient or tumor which expresses these genes normally. It does no good to produce cytokines around a tumor which has developed antigenic diversity or disguise and will therefore be unrecognized by the immuno-effector cells called to the site. It would be of little benefit to block the action of the Ras oncoprotein if RAS over-expression is not taking place in that particular cell. Methods for characterization of cancers are as known to the art and as described herein.
In the method of this invention for expressing a protein in a cell containing androgen receptors, the cell, which may be normal or cancerous, is contacted with a nucleic acid complex, preferably a DNA complex, of a compound comprising a steroid moiety capable of binding to an androgen receptor of said cell, said steroid moiety being covalently linked to a polycationic salt having a pharmaceutically acceptable counterion. The protein is detectably expressed in the cell. Expression is detected by means known to the art, e.g. as described herein.
Preferably a subject in whom expression is desired is treated with the nucleic acid complexes of this invention by injection of a water-soluble preparation of a complex containing DNA encoding a therapeutic protein as discussed above. Suitable pharmaceutical carriers for such injections include normal saline, 5% glucose, and other carriers known to the art.
The treatments of this invention are especially useful for patients who have been treated by androgen ablation since androgen deprivation increases the transcription, number and receptivity of the patient""s androgen receptors.
When the complexes of this invention are used for diagnosis, they comprise DNA or other substances labelled so as to be detectable in diagnostic methods, e.g. by radiolabelling, such as with Tc99. The presence of the radio-labelled substances is detected using radiation detectors known to the art. The presence of such substances in tissue such as kidney tissue, lung tissue, etc. not normally having androgen receptors indicates the presence of metastatic sites.